The long-term goals of this work are to understand cellular and molecular mechanisms of pancreas development during vertebrate embryogenesis. Over the past 2.5 years, we have conducted ascreen.for genes regulating pancreas development using a transgenic zebrafish line expressing GFP in the endoderm and endoderm-derived organs. We have also developed wholemount immunostaining techniques and utilized novel transgenic lines to characterize these mutants. Importantly, we carried out a detailed analysis of wild-type pancreas development in zebrafish (Field et al., 2003b), a study that has facilitated the use of the zebrafish system to identify and/or test genes implicated in this process. Specifically, we found that the zebrafish pancreas is formed by the fusion of two buds, a posterior one that contains only islet cells and an anterior one that gives rise to the exocrine cells and the pancreatic duct. The pancreatic duct in turn gives rise to new islet cells. This initial separation of endocrine and exocrine lineages should facilitate their investigation. We propose to continue our studies with the following specific aims. 1) Analyze the pancreas mutants identified and recovered from our screen. 2) Further characterize 6 mutants including detailed phenotypic analysis and isolation of the affected gene. 3) Analyze the role of the surrounding tissues in pancreas development and beta cell differentiation. We will analyze in detail the emergence of the initial islet cells (those that form the posterior bud) with the goals of identifying the signals that regulate this process. This information will be useful for designing cell culture protocols to differentiate stem cells into beta cells. We will also test the hypotheses that signals from the lateral plate mesoderm control the development of the anterior pancreatic bud, as well as the emergence of new islet cells from the pancreatic duct. 4) Continue to develop reagents and techniques to facilitate the use of the zebrafish as a rapid screening tool for genes regulating pancreas development and beta cell differentiation.